The complex role of the cytoskeleton in the intracellular traffic of cholesterol from droplet to mitochondrion involves not only microfilaments and microtubules (as we have previously shown), but also involves the participation of intermediate filaments, as we now show for the first time. The disruption of intermediate filaments with acrylamide enhances steroidogenesis as efficiently as ACTH or colchicine although each has a different time course. These findings confirm the model that polymerized filaments can act as a barrier of cholesterol processing in steroid- producing cells.